The skin is the largest organ in the human body and functions as a protective barrier against the agents, such as infectious agents, in the external environment, and as a waterproof barrier that maintains fluid homeostasis and prevents evaporation of tissue moisture. Intact skin prevents local infection of the dermis or other underlying tissue with microorganisms. If unchecked, such local infections can become invasive and can result in sepsis or systemic infection.
The temporary loss of skin often results in mortality or morbidity. Widespread loss of skin integrity is most often associated with major burns. The importance of the barrier function of skin is demonstrated by the fact that for a given age, mortality is directly related to burn size. Current standards of care recommend resurfacing of the patient as soon as possible to restore fluid homeostasis and barrier function.
Burn patients are most often resurfaced with autologous skin grafts. Autologous skin grafts techniques are widely used, but frequently fail. For example, a patient with large burns may not have sufficient donor skin available to cover the recipient site.
Two major alternative patient salvage techniques are currently available. One is to completely excise all the burned tissue and cover the patient with a temporary epidermis. INTEGRA (Johnson and Johnson, New Brunswick, N.J.) is a neo-dermis bilayer having an inner layer of bovine collagen and chondroitin-6-sulfate and an outer layer of silicone. After neo-dermal angiogenesis 14 to 21 days later, the silicone outer layer is surgically removed and the patient is resurfaced with thin, widely meshed autografts. The thin autografts offer the distinct advantage of allowing limited donor sites to heal and be serially harvested. Although the burn community has embraced INTEGRA, the susceptibility to infection has limited its use in patients with dirty or infected burn wounds (1). The other major limiting factor has been donor site availability. Patients with catastrophic burn injury may not have sufficient donor site available despite the obvious advantage of requiring much thinner autografts.
The second commonly employed technique is to excise the burn wound and to provide a temporary dermal/epidermal cover of cadaver skin to restore the skin's barrier function. Autologous keratinocytes are harvested and placed into culture. EPICEL (Genzyme Corporation, Cambridge, Mass.), the only commercially available permanent skin replacement, forms keratinocytes 2 to 6 cell layers thick attached to fine mesh gauze. These autografts are available in 3 to 4 weeks from the time of biopsy. In the case of large burns, this timetable often coincides with development of burn wound sepsis. Bacterial contamination and other factors cause high failure rates of EPICEL that are not seen with traditional split-thickness skin grafts (2).
Further advances in burn wound care may come from the arena of tissue engineering. One proposal has been to restore the barrier function with chimeric cultures of autologous keratinocytes mixed with established allogeneic cell cultures (3, 4). This could permit earlier wound coverage by significantly decreasing the time required for definitive culture formation. Slower permanent resurfacing of the skin would occur with autologous keratinocytes as the allogeneic cells are rejected. One major obstacle is the need for an established, cultured, and tested allogeneic keratinocyte line. This has heretofore been impractical as keratinocytes senesce in culture and new keratinocyte lines would have to be continually established and tested.
Skin is composed of two layers, the dermis and the epidermis. The dermis is a connective tissue containing fibroblasts embedded in a matrix of collagen and elastic fibers. The epidermis, in contrast, consists primarily of cells with little connective tissue.
Keratinocytes are the major cellular component of the epidermis and comprise about 80% of the cells in adult human skin. They are the epidermal component responsible for providing the skin's barrier, reparative, and regenerative properties. Their name derives from their predominant cytoskeletal component, keratins. Keratins are the subunits of keratin filaments and are divided into two types: type I (acidic) keratins and type II (basic or neutral) keratins. All epithelia express type I and type II keratins, which range in molecular weight from 40 kDa to 70 kDa. Different epithelial tissues express specific pairs of keratins. Heterodimers of type I and II keratins form intermediate filaments that confer tensile strength and structural support to the cells and resulting epithelial tissue.
The epidermis consists of four morphologically and biochemically distinct layers. The basal layer of keratinocytes is in contact with the basement membrane, which separates the epidermis from the dermis. Basal cells are the only keratinocytes in intact skin capable of mitosis and, as such, are the source of all other keratinocytes in the epidermis. They attach to the substratum through hemidesmosomes and to adjacent cells through desmosomes and adherens junctions (reviewed in Jensen and Wheelock, 1996). Basal layer cells are columnar in shape and produce keratins K5 and K14.
The first suprabasal keratinocyte layer is the stratum spinosum, so named for the “spiny” appearance of the many desmosomal contacts between adjacent cells. Keratinocytes in this layer no longer produce K5 and K14 but, instead, synthesize differentiation-specific keratins K1 and K10. Cells begin to produce involucrin and epidermis-specific transglutaminases in the upper stratum spinosum. Morphologically, spinous cells are larger and more flattened than basal cells (reviewed in Holbrook, 1994).
Involucrin expression is localized to the cytoplasm of suprabasal and granular cells (Mansbridge and Knapp, 1987; Murphy et al., 1984) of normal skin. Involucrin has also been shown to localize pericellularly in these layers, but tissues used for these immunohistochemistry experiments were not fixed, and it was hypothesized that the protein diffused to the cell borders during or after the sectioning process (Smola et al., 1993; Watt, 1983).
Transglutaminases are a family of calcium-dependent enzymes that catalyze the covalent cross-linking of proteins to each other or to polyamines (reviewed in Rice et al., 1994). The keratinocyte transglutaminase isozyme, TGK, is membrane-bound in suprabasal epidermis and catalyzes the cross-linking of involucrin and at least six other membranous proteins to form the cornified envelope (CE) (reviewed in Rice et al., 1994). The CE is a highly stable insoluble protein structure formed beneath the plasma membrane that is resistant to detergents and reducing agents and confers strength and rigidity to the terminally differentiated cells of the uppermost epidermal layer. Many CE components, including TGK, are synthesized in the stratum spinosum although the envelope is not formed until the cells transition from the stratum granulosum to the stratum corneum. TGK is also found in all subsequent layers of the epidermis (Michel et al., 1997; Mansbridge et al., 1987; Asselineau et al., 1989). The enzyme is inactive until, in the final stage of differentiation, a loss of membrane integrity results in an influx of calcium into the cell (Aeschlimann and Paulsson, 1994).
As keratinocytes differentiate further, they form the stratum granulosum. Cells of this layer are characterized by distinct electron-dense keratohyalin granules containing profilaggrin, the protein precursor of filaggrin (reviewed in Dale et al., 1994). Granular cells also contain lipid-filled granules that, during the transition zone between the stratum granulosum and stratum corneum, fuse with the plasma membrane and release their contents into the extracellular space, conferring hydrophobicity to the epidermal surface.
As the differentiating keratinocytes transition from the granular to the cornified layer, the profilaggrin is cleaved to yield filaggrin, which is involved in the alignment and aggregation via disulfide bonds of keratin bundles called macrofibrils (reviewed in Holbrook, 1994). Macrofibrils are the basic structural unit of the cornified envelope. In normal skin sections, filaggrin is localized in the granular layer, with some faint, continued staining in the cornified sheets (Michel et al., 1997; Asselineau et al., 1989; Mansbridge et al., 1987). It should be noted that antibodies against filaggrin detect profilaggrin as well as its cleavage products, which accounts for the strong, punctate staining pattern of the keratohyalin granules of the stratum granulosum.
The uppermost epidermal layer is the stratum corneum. Cells of this layer, having completed the differentiation process, have lost their nucleus and all metabolic function. They consist primarily of keratin filaments encased by the now-complete CE and overlying plasma membrane. Corneal cells are joined together by modified desmosomes and are ultimately sloughed off in sheets from the skin's surface.
Keratinocytes also produce cadherin adhesion molecules. The classical cadherins, N-, E-, and P-cadherin, are a subfamily of cadherins that localize to the adherens junctions and mediate cell-cell adhesion through homotypic interactions. These calcium-dependent, transmembrane glycoproteins play major regulatory roles in tissue formation and facilitate intercellular interactions. Cadherin complexes are also thought to participate in the transduction of intracellular signals through their association with the actin cytoskeleton (Knudsen et al., 1998). Keratinocytes produce both E- and P-cadherins. E-cadherin is found in all living layers of the epidermis (reviewed in Jensen and Wheelock, 1996) while P-cadherin is found in the stratum basale and immediately suprabasal cells.
The skin regenerates every 28 days (reviewed in Sams, 1996). As basal keratinocytes lose contact with the basement membrane they produce daughter cells that terminally differentiate as they move upward through the suprabasal layers to the skin surface. Terminal differentiation involves a series of biochemical and morphologic changes that result in a layer of dead, flattened squames that carries out the barrier and protective functions of the skin. These cornified cells are routinely sloughed off and replaced with newly differentiated cells, maintaining the controlled balance between proliferation and differentiation involved in tissue homeostasis.
Keratinocyte Culture
Cultivated cells isolated from disaggregated human skin have been used for over two decades to study keratinocyte growth and differentiation (reviewed in Leigh et al., 1994). Human foreskin keratinocytes cultured in the presence of a 3T3 mouse embryo fibroblast feeder layer or in serum-free medium formulations exhibit sustained growth for approximately 80 population doublings prior to senescence (reviewed in Leigh and Watt, 1994). Human keratinocytes cultured under these conditions can express differentiation-specific proteins, such as involucrin and keratins K1 and K10, in a position-specific manner (reviewed in Fuchs and Weber, 1994).
Although features of squamous differentiation and limited stratification are consistently observed in cultured keratinocyte monolayers, normal tissue architecture is not evident. The discovery that epidermal cells in traditional submerged culture grow optimally when plated on top of non-proliferating fibroblasts was a significant contribution to the study of keratinocyte cell biology (Rheinwald, 1980; reviewed in Fuchs, 1993). The use of this culturing system allowed investigators to serially cultivate keratinocytes for a wide range of purposes. Unfortunately, the submerged culture system allows for limited, aberrant stratification consisting of only a few layers of keratinocytes, which lack the specific morphological and biochemical characteristics of a true stratified epithelium. For example, several markers of normal epidermal differentiation are not produced in submerged culture, such as keratins 1 and 10 and the late stage marker, filaggrin (reviewed in Fuchs, 1993). Several factors limit this in vitro system. First, in vivo epidermis sits atop the dermis and receives its nutrients and growth signals via diffusion from the dermis through the basement membrane to the overlying basal cells. This imparts a polarity to the tissue that cannot be achieved in traditional submerged cell cultures that are fed through all upper surfaces to the bathing medium. Second, epithelial cells grown in this manner do not produce a basement membrane and lack exposure to its mesenchymal cues for normal differentiation and growth (reviewed in Fusenig, 1994). Third, while the fibroblast “feeder” layer promotes keratinocyte proliferation, the cultivation of cells in this manner results in the same aberrant or absent differentiation characteristics as is seen in cultures grown on a plastic substratum. This indicates that keratinocytes require a more complex mesenchyme of fibroblasts and extracellular matrix proteins to produce a functional epidermis. Accordingly, the traditional submerged culture system is appropriate only for relatively simplistic studies.
Several systems developed and tested over the past 20 years are designed to develop more in vivo-like keratinocyte culturing conditions. In 1979, collagen gels were first used as physiologic “rafts” upon which to grow keratinocytes at the air-medium interface (Bell et al., 1979; reviewed in Fusenig, 1994; Parenteau et al., 1992). This allowed the nutrients and growth factors from the medium to diffuse through the collagen to the basal layer of keratinocytes and exposed the uppermost keratinocyte layers to the air. These added in vivo-like conditions improved the histological architecture of the cultured epidermis. Full stratification and histological differentiation can be achieved using these three-dimensional “organotypic” culture methods, which have been continually modified to more closely recapitulate the in vivo growth environment.
In later organotypic systems, live fibroblasts were incorporated into collagen gels, keratinocytes were placed atop contracted collagen “rafts”, and the entire raft was lifted to the air-medium interface (reviewed in Fusenig, 1994) to emulate intact skin, where resident dermal cells such as dermal fibroblasts are involved in signaling keratinocytes to produce a basement membrane and an epithelium with a significantly improved differentiation program (reviewed in Watt and Hertle, 1994). The viable fibroblasts provide valuable signals and promoted production by cultured keratinocytes of basement membrane proteins.
Keratinocyte Differentiation
In both intact skin and in organotypic culture, differentiating keratinocytes produce proteins unique to particular differentiation stages. The presence and localization of these protein markers can be detected using biochemical and immunohistochemical methods and used to determine whether an epithelial tissue is differentiating (stratifying) normally. The expression profiles of several such proteins are presented below.
The most well-studied proteins for determining epidermal differentiation are the keratins, most notably K5/14 and K1/10, which make up the intermediate filament network in epidermal keratinocytes. This network provides a cellular framework that extends from the nucleus to specific adhesion junctions called desmosomes and hemidesmosomes (reviewed in Fuchs and Cleveland, 1998). These cell-cell and cell-substratum interactions, when connected to keratin filaments, are responsible for anchoring keratinocytes to each other and to the underlying basement membrane. The K5/K14 pair of keratin filaments is expressed in the basal cells of stratified squamous epithelia (reviewed in Fuchs, 1993). As expected, K14 mRNA is present only in the basal layer of normal human epidermis (Stoler et al., 1988). As basal cells of the epidermis begin to differentiate, they downregulate their expression of K5/14 and begin to produce differentiation-specific keratins. The K1/K10 pair of keratin filaments is expressed in the suprabasal layers of the epidermis (reviewed in Fuchs, 1993). K1 and K10 are early markers of terminal differentiation as they are produced by keratinocytes upon leaving the basal layer. In samples of intact human skin, K1 is from the first suprabasal layer throughout the surface of the tissue (Stoler et al., 1988; Stark et al., 1999; Asselineau et al., 1989; Boukamp et al., 1990).
In primary human keratinocyte organotypic cultures, initiation of K1 protein synthesis is delayed relative to intact skin. Protein localization begins 5-8 cell layers above the basement membrane as opposed to 1-2 layers in normal skin samples. Induction of K1 production normalizes following transplantation of the organotypic cultures onto nude mice (Smola et al., 1993; Stark et al., 1999). These studies did not examine K1 mRNA expression.
Involucrin is localized to the suprabasal cell membranes in primary keratinocyte organotypic cultures, even in fixed tissue (Boukamp et al., 1990; Smola et al., 1993; Stark et al., 1999; Watt et al., 1987), and several groups also found that the membranous pattern remained after transplantation onto nude mice (Breitkreutz et al., 1997; Watt et al., 1987).
TGK expression seems to vary in organotypic cultures of normal human keratinocytes, first appearing either in the stratum spinosum or the stratum granulosum and continuing up through the stratum corneum (Michel et al., 1997; Stark et al., 1999).
Organotypic cultures of normal human keratinocytes also display localization of filaggrin in the granules of the uppermost strata of the epidermal tissue (Boukamp et al., 1990; Michel et al., 1997; Stark et al., 1999).
In addition to differentiation markers, one can also assess the structural and functional integrity of an epithelial tissue by monitoring for the presence and localization of adhesion proteins.
To date, neither of E- nor P-cadherin has been examined in organotypic culture of primary keratinocytes, although E-cadherin has been detected immunohistochemically in normal skin (Haftek et al., 1996).
Smola and coworkers (Smola et al., 1998) have clearly shown that organotypic cultures composed of normal dermal fibroblasts and keratinocytes develop a basement membrane zone capable of supporting cell type specific adhesion structures such as hemidesmosomes.